Many methods are available for conjugating oligonucleotides with other molecules. These methods typically involve attachment of a reactive moiety on the target entity to be coupled with the oligonucleotide. The target entities with the reactive moieties are often made separately, usually by organic synthesis methods, and purified before use.
In each case the oligonucleotides are modified with appropriate functional groups for reacting with the reactive moieties on the target entities. Modifications of oligonucleotides are often accomplished by making special phosphoramidites and/or modified bases, and incorporating them into the oligonucleotide sequences at the desired points. Many of these amidite reagents contain functional groups that require protecting groups for the coupling of these reagents to the oligonucleotides. These protecting groups must be removed before the subsequent conjugation reaction occurs. Alternatively, the reactive moieties on the target entities may be created before the conjugation to the oligonucleotides.
Most of current coupling chemistries involve the use or creation of hydrolytically and/or oxidatively unstable species of at least one of the conjugation partners. This is a problem under conditions (typically in aqueous solutions) needed for conjugation with the oligonucleotides (or protein, or any organic insoluble/water soluble species). Newer conjugation chemistries may generate a novel structure upon reaction. For example, U.S. Pat. No. 6,737,236, issued to Pieken et al., discloses cycloaddition reactions for the conjugations of biomolecules. One example, a 1,3-dipolar cycloaddition conjugation between an alkyne and an azide (later labeled under the general term “Click” chemistry by Sharpless et. al. Angew. Chem. Int. Ed. 40: 2004 (2001) produces a substituted triazine as part of the conjugation product. These new chemical entities can be a problem if the oligonucleotide conjugates are used in humans because these new chemical entities may cause toxicity unrelated to the oligonucleotide products.